This study demonstrates the feasibility and efficacy of using flow cytometric analysis with intracellular cytokine staining for characterization of T-cell phenotype and functional status in extensive alopecia areata (EAA) scalp skin. Cell suspensions were made from scalp punch biopsies taken from 12 patients with long-standing EAA (average disease duration 14 years, 95% hair loss) and six control subjects. EAA samples had a lower percentage of CD-3-expressing cells, but CD-4/CD-8 ratios remained similar to controls. Expression of CD-69 was found only in EAA scalp biopsies, suggesting that T-cells from EAA scalp have undergone activation. No difference was found in tumor necrosis factor alpha expression. Surprisingly, EAA scalp T-cells produced less IL-2 and CD-8 T-cells produced less IFN-gamma. Immunohistochemical staining of formalin-fixed paraffin-embedded specimens demonstrated that IFN-gamma-producing cells in EAA scalp were not greater in number than in normal specimens. The few identified IFN-gamma-producing cells demonstrated no tendency to localize to the perifollicular region, and were similarly distributed as in control specimens. The abnormalities in cytokine production may explain the relative paucity of inflammatory change observed in the clinical setting and suggest that T-cell responses in EAA scalp are tightly, albeit aberrantly, regulated via mechanisms of peripheral T-cell tolerance.